The presence of elevated numbers of activated mast cells (MCs) in the joints of patients with rheumatoid arthritis (RA) raised the possibility in the 1980s that MCs participate in the inflammation, proteolytic damage, and/or remodeling of the arthritic joint. Using MC-deficient mice, we and others subsequently demonstrated that these immune cells do indeed contribute substantially to experimental inflammatory arthritis. Nevertheless, the relevant mediators produced by the Fc3RIII- and C5a-activated MCs in the synovium that adversely affect the joint had not been identified. Mouse MC protease (mMCP) 6 and mMCP-7 are members of the chromosome 17A3.3 family of tryptic proteases that are preferentially stored in the secretory granules of MCs as homotypic and heterotypic tetramers ionically bound to heparin-containing serglycin proteoglycans. The human ortholog of mMCP-6 is hTryptase-21. We generated enzymatically active mMCP-6, mMCP-7, and hTryptase-21. We also recently created inbred transgenic C57BL/6 mouse strains that differ in their expression of mMCP-6, mMCP-7, and heparin. Using the latter mice, we showed that C57BL/6 mice that lack tryptase7heparin complexes have reduced inflammation and diminished loss of aggrecan proteoglycans from their cartilage in two different mouse models of inflammatory arthritis. The overall objectives of the proposed studies are to use complementary approaches to determine at the molecular level the roles of mouse and human tetramer-forming tryptases in RA and mouse models of this autoimmune disorder. Our genetically modified mouse strains, recombinant tryptases, and other key protease-specific reagents will be used in the proposed studies to determine how MC-restricted tetramer-forming human and mouse tryptases affect fibroblast-like synoviocytes (FLS) and chondrocytes at multiple levels in varied in vitro and in vivo studies, as well as how these mesenchymal cells affect MCs. Additional studies will be carried out to determine how exocytosed mMCP-6 and hTryptase-21 are negatively regulated in the arthritic joint, and even in MCs themselves. The factors and mechanisms that control the expression of enzymatically active tryptases inside MCs have not been identified. To this end, we recently discovered that FLS-derived IL-33 induces MCs to increase their expression of tryptases at the mRNA and protein levels. Thus, additional mechanistic studies are proposed to understand how IL-33 controls the transcription of the mMCP-6 and hTryptase-21 genes in MCs and/or the stability of their transcripts. In combination, these studies promise to provide novel insights into both MC and synovial physiology, as well as elucidate novel disease mechanisms in human inflammatory arthritis.